Cytometer sperm sex sensing apparatus with an avalanche photodiode

ABSTRACT

A cytometer includes an avalanche photodiode, a switching power supply, a filter, and voltage adjustment circuitry. The switching power supply includes a feedback loop. The filter is electrically connected between the switching power supply and the avalanche photodiode. The voltage adjustment circuitry adjusts a voltage on the feedback loop based at least in part on a voltage measured between the filter and the avalanche photodiode.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/056,003, filed on Aug. 6, 2018, which is a continuation of Ser. No.15/839,073, filed on Dec. 12, 2017, which claims the benefit of U.S.Provisional Patent Application No. 62/559,336, filed on Sep. 15, 2017,the entireties of which are herein incorporated by reference.

BACKGROUND

Generally, this application relates to flow cytometry. In particular,this application relates to techniques for implementing an avalanchephotodiode in a cytometer to detect the sex of a sperm cell.

Flow cytometry may utilize light detection to assess characteristics ofparticles, such as cells, flowing through the cytometer. In certainapplications, a cytometer may detect light emitted by cells, includinglight emitted by fluorescent, DNA-intercalating dye. The ability todetect such emitted light may permit accurate and sensitivedifferentiation of certain characteristics of the cells.

One such application is the determination of whether a sperm cell hastwo X-chromosomes (which may produce a female zygote) or alternativelyan X-chromosome and a Y-chromosome (which may produce a male zygote). Asperm cell with two X-chromosomes may have approximately 3% more DNAthan a sperm cell with an X-chromosome and a Y-chromosome. Byidentifying the chromosomal content of a sperm cell, it may be possibleto create a relatively high-purity population of XX or XY sperm cells.Such a population may be generated, for example, by keeping the desiredgender and killing the undesired one (or by segregating the twogenders). The substantially high-purity population may be used toinseminate a female animal (such as bovids, equids, ovids, goats, swine,dogs, cats, camels, elephants, oxen, buffalo, or the like) to obtain adesired male or female offspring, with relatively high probability.

The characteristics of the chromosomes may be identified by staining theDNA of sperm cells with a fluorochrome (for example, a DNA-intercalatingdye). The stained sperm cells may be forced to flow in a narrow streamor band and pass by an excitation or irradiation source such as a laserbeam. As the stained sperm cells (a plurality of particles) areirradiated, the fluorochrome in the plurality of particles emits aresponsive fluorescent light. The amount of fluorescent light may varybased at least in part on a relative amount of at least one particledifferentiation characteristic (for example, the relative amount ofchromosomes) present in each of the plurality of particles. Thefluorescent light may be received by one or more optical elements thatultimately focus the received light onto a detection component, such asa photomultiplier tube (“PMT”). The detection component may generate anelectrical, analog signal in response to the received light. The analogsignal may vary in correspondence with the amount of received light.This signal may then be processed (for example, digitized and analyzedby a processor) to assess the chromosomal content of the sperm cell.

One consideration when using a flow cytometer to perform this type ofdifferentiation may be the sensitivity of the light-sensing componentryof the cytometer. The amount of light emitted (that is, the number ofphotons emitted) by a single cell that has been stained with afluorescent, DNA-intercalating dye may be relatively low. As a result,the detection componentry must be sufficiently sensitive, both in orderto make the detection, and even more so in order to detect differencesin certain characteristics between different cells.

Cytometer technology may implement a PMT to achieve sufficientsensitivity for an application such as determining the chromosomalcontent of sperm cells. The PMT's relatively large optical signal gains(for example, 10̂7 or greater) enable this particular application. Forexample, PMTs with such optical gain may be capable of sensing asufficiently low level of light emission, and of discriminating theroughly 3% difference in total fluorescence between stainedXX-chromosome and XY-chromosome bearing sperm cells. However, PMTs arerelatively expensive. They may also require a relatively long period oftime to “warm up” (during which the PMT should not be exposed to light)once the flow cytometer has been turned on. Additionally a PMT mayrequire voltage in the thousands of volts. Furthermore, a PMT mayrequire a shutter to block light when not in use, as normal roomlighting can damage and break the PMT if exposed.

SUMMARY

According to certain inventive techniques, a cytometer includes anavalanche photodiode, a switching power supply, a filter, and voltageadjustment circuitry. The switching power supply includes a feedbackloop. The filter is electrically connected between the switching powersupply and the avalanche photodiode. The voltage adjustment circuitryadjusts a voltage on the feedback loop based at least in part on avoltage measured between the filter and the avalanche photodiode.

The cytometer may further include a temperature sensor configured tosense a temperature and generate a corresponding temperature signalencoding temperature data, wherein the voltage adjustment circuitry isfurther configured to adjust the voltage on the feedback loop based atleast in part on the temperature data and the voltage measured betweenthe filter and the avalanche photodiode. The voltage adjustmentcircuitry may further be configured to adjust the voltage on thefeedback loop based at least in part on at least one characteristic ofthe avalanche photodiode, the temperature data, and the voltage measuredbetween the filter and the avalanche photodiode. The voltage adjustmentcircuitry may further be configured to adjust the voltage on thefeedback loop based at least in part on at least one characteristic ofthe avalanche photodiode and the voltage measured between the filter andthe avalanche photodiode. The voltage adjustment circuitry may include:an analog-to-digital converter configured to convert the voltagemeasured between the filter and the avalanche photodiode into a digitalmeasured signal encoding measured voltage data; a processor configuredto process at least the measured voltage data to generate a digitaladjustment signal; and a digital-to-analog converter configured toconvert the digital adjustment signal to an adjustment voltage, whereinthe adjustment voltage influences the voltage on the feedback loop. Theprocessor may be configured to process at least temperature data and themeasured voltage data to generate the digital adjustment signal. Theprocessor may be configured to process at least temperature data, datacorresponding to at least one characteristic of the avalanchephotodiode, and the measured voltage data to generate the digitaladjustment signal. The at least one characteristic of the avalanchephotodiode may include at least one of a breakdown voltage and a reversebias voltage corresponding to a predetermined optical gain. Thecytometer may further include: a first amplifier configured to amplify avoltage at an anode of the avalanche photodiode to form a firstamplified voltage; and a second amplifier configured to amplify thefirst amplified voltage to generate a second amplified voltage. Thepower supply may include a DC/DC power supply. The avalanche photodiodemay be: arranged to receive an amount of fluorescent light emitted byeach of a plurality of particles; the amount of received fluorescentlight may vary based at least in part upon a relative amount of at leastone particle differentiation characteristic present in each of theplurality of particles; and the avalanche photodiode may be configuredto convert the amount of received fluorescent light into at least onesignal which varies based upon the amount of received fluorescent light.

According to certain inventive techniques, a method includes: receivingan input voltage at input circuitry of a power supply; transforming,with a transformer, a voltage supplied by the input circuitry into atransformed voltage; receiving, at output circuitry of the power supply,the transformed voltage; generating, by the output circuitry, an outputvoltage; feeding back a feedback voltage corresponding to the outputvoltage to the input circuitry; receiving the output voltage at afilter; generating, by the filter, a filtered voltage; receiving thefiltered voltage at an avalanche photodiode; receiving the filteredvoltage at voltage adjustment circuitry; and adjusting the feedbackvoltage by the voltage adjustment circuitry according to at least thefiltered voltage. The method may further include generating, by atemperature sensor, a temperature signal corresponding to a measuredtemperature, wherein said adjusting the feedback voltage furthercomprises adjusting the feedback voltage by the voltage adjustmentcircuitry according to at least the filtered voltage and the measuredtemperature. Said adjusting the feedback voltage further may includeadjusting the feedback voltage by the voltage adjustment circuitryaccording to at least the filtered voltage, the measured temperature,and at least one value corresponding to a characteristic of theavalanche photodiode. Said adjusting the feedback voltage further mayinclude adjusting the feedback voltage by the voltage adjustmentcircuitry according to at least the filtered voltage and at least onevalue corresponding to a characteristic of the avalanche photodiode.Said adjusting the feedback voltage may further include: converting,with an analog-to-digital converter, the filtered voltage into a digitalmeasured signal encoding filtered voltage data; processing, with aprocessor, at least the filtered voltage data to generate a digitaladjustment signal; converting, by a digital-to-analog converter, thedigital adjustment signal to an adjustment voltage; and adjusting thefeedback voltage according to the adjustment voltage. Said processing atleast the filtered voltage data may further include processing at leasttemperature data and the filtered voltage data to generate the digitaladjustment signal. Said processing at least the filtered voltage datamay further include processing at least data corresponding to at leastone characteristic of the avalanche photodiode, temperature data, andthe filtered voltage data to generate the digital adjustment signal. Theat least one characteristic of the avalanche photodiode may include atleast one of a breakdown voltage and a reverse bias voltagecorresponding to a predetermined optical gain. The method may furtherinclude: amplifying, with a first amplifier, a voltage at an anode ofthe avalanche photodiode to form a first amplified voltage; andamplifying, with a second amplifier, the first amplified voltage togenerate a second amplified voltage. The input circuitry, thetransformer, and the output circuitry may comprise a DC/DC power supply.

According to certain inventive techniques, a flow cytometry apparatusincludes: a flow chamber configured to direct a fluid stream includingsample particles through a particle interrogation location; a laserconfigured to emit electromagnetic radiation along a beam path to theparticle interrogation location; an avalanche photodiode configured to:receive electromagnetic radiation from the interrogation location; andoutput a time-varying analog signal indicative of an intensity of thereceived electromagnetic radiation; at least one amplifier configured toamplify the time-varying analog signal; an analog-to-digital converterconfigured to receive the amplified time-varying analog signal andproduce a corresponding digitized output signal; and a processorconfigured to analyze the digitized output signal.

According to certain inventive techniques, a method for analyzing spermcells contained in a fluid stream as the sperm cells flow through aninterrogation location includes: emitting electromagnetic radiation froma laser; illuminating, with the electromagnetic radiation emitted by thelaser, the fluid stream and the sperm cells contained therein;detecting, with an avalanche photodiode, electromagnetic radiationemitted from the interrogation location; generating, by the avalanchephotodiode, a time-varying analog signal indicative of an intensity ofthe detected electromagnetic radiation; converting, with ananalog-to-digital converter, the time-varying analog signal into acorresponding digital signal; and analyzing, by a processor, the digitalsignal to determine characteristics of the sperm cells in the fluidstream.

According to certain inventive techniques, a method for assessing anamount of DNA within a nucleus of a sperm cell includes: staining theDNA within the nucleus of the sperm cell; irradiating the stained DNAwithin the nucleus of the sperm cell; and detecting, with an avalanchephotodiode, fluorescent light emitted from the irradiated and stainedDNA within the nucleus of the sperm cell. The method may further includedifferentiating X chromosome bearing sperm cells and Y chromosomebearing sperm cells by: determining a sex of a sperm cell using thedetected amount of DNA within the nucleus of the sperm cell; anddifferentiating between a plurality of sperm cells based upon said sexdetermination. The characteristics of the sperm cells may includecorresponding amounts of DNA within the nuclei of the sperm cells. Themethod may further include deactivating a given sperm cell based uponthe determined amount of DNA within the nucleus of the given sperm cell.Said deactivating may include photo-damaging the given sperm cell. Themethod may further include: forming droplets a plurality having one ofsaid sperm cells entrained; charging each of said dropletsdifferentially based upon said sex differentiation characteristic ofsaid sperm cells entrained in said droplets; deflecting each of saiddroplets; and differentially collecting each of said droplets based uponsaid sex differentiation characteristic of said sperm cells entrained insaid droplets.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates a flow cytometer, according to certain inventivetechniques.

FIG. 2 shows a block diagram for circuitry capable of capable ofdetecting the chromosomal content of sperm cells using an avalanchephotodiode, according to certain inventive techniques.

FIG. 3 illustrates a flowchart for a method, according to certaininventive techniques.

FIG. 4 illustrates a flowchart for a method, according to certaininventive techniques.

FIG. 5 illustrates a flowchart for a method, according to certaininventive techniques.

FIGS. 6A-6X depict circuit schematics for a portion of a cytometer,according to certain inventive techniques.

The foregoing summary, as well as the following detailed description ofcertain techniques of the present application, will be better understoodwhen read in conjunction with the appended drawings. For the purposes ofillustration, certain techniques are shown in the drawings. It should beunderstood, however, that the claims are not limited to the arrangementsand instrumentality shown in the attached drawings.

DETAILED DESCRIPTION

Certain inventive techniques discussed herein describe a cytometer thatuses an avalanche photodiode (“APD”) with a lower cost implementationinstead of a PMT to obtain low-level fluorescence detection, such thatbull sperm chromosomal content can be determined or differentiated. Theadvantageous techniques may also enable the use of an APD in othercytometer sensing applications. An APD has substantial advantages over aPMT, including, for example, lower cost, no “warm-up” period, smallerand more efficient active area, improved control over the detectioncomponent, lower bias voltage (hundreds of volts vs. thousands of voltsfor a PMT), and no need for a light-blocking shutter when not in use.

With regard to the active area, a PMT may have a relatively large activearea (in one example, approximately 13.7 mm×3.9 mm) whereas an exemplaryAPD may have a substantially smaller and more symmetrical active area(in one example, a circular area having a 1 mm diameter). This mayresult in an improved fill factor for the sensor (for example, one orderof magnitude or greater). By improving fill factor, it is easier toalign the APD (versus the PMT) with the laser beam cross-sectional area,which makes the optical alignment relatively easier when constructing asystem.

With regard to improved control over the APD (versus the PMT), certaininventive techniques described herein allow for improved compensationand biasing, for example, by incorporating the APD into the cytometer ina manner that leverages the APDs quantum efficiency combined with thesensor optical gain. The quantum efficiency of the APD is related to thecytometer through a chosen dye and optical excitation wavelength. Theoptical gain factor of the APD may combine with the quantum efficiency,which is cell-stain (dye) dependent to produce the sensor output signal.

According to certain inventive techniques, the APD is configured as anelement of the voltage supply output filter network. Furthermore, thecytometer may include temperature compensation circuitry. APD opticalgain may vary with temperature. If the temperature change is known, thebias voltage on the APD may be adjusted to compensate for thetemperature change (to keep gain substantially constant). Alternatively,a heater that keeps the APD at constant temperature, or a relativelygiant thermal mass, or a thermal electric cooler could also be used tostabilize the APD temperature (and thus keep the APD optical gainsubstantially constant).

According to certain inventive techniques, APD conditioning circuitrymay employ a two-stage feedback network to achieve relatively low noiseperformance. The first stage may be an analog stage controlled by aresistive feedback circuit to a flyback converter in a high-voltagepower supply that reverse-biases the APD. The resistive feedback circuitin the power supply may have a digital-to-analog converter connectedthrough circuitry that provides the ability to change the output voltageof the flyback converter. The second stage may allow the APD to become apart of an output filter network of the high-voltage power supply. Thesecond stage may be designed to take into consideration that the flybackconverter feedback may not accurately represent the voltage at the APDdue to relatively large output filtering used to achieve low noiseperformance. The second stage feedback network may measure the outputvoltage after the first output filter stage and digitize it, becoming adigital feedback element in the control loop. This configuration maycombine both analog and digital feedback elements in a hybridizedimplementation. By using the second stage feedback network, relativelylarge values in the output filtering, such as relatively large polecompensation generating relatively large resistor and capacitorcombinations, may be able to be used for improved noise filteringthrough the use of both the analog and digital feedback mechanisms.

The second stage feedback network may operate by sensing the voltageafter a first output filter stage of the high-voltage power supply. Thismay take into consideration the voltage substantially near or at theAPD. The sensed voltage may be digitized and processed by a processor(for example, a single processor or a plurality of processors orprocessing elements working together) in a substantially real-timemanner. The processor may evaluate the sensed voltage proximate the APD(after the first output filter) as well as, optionally, other parameterssuch as the APD temperature and characteristics particular to a givenAPD (for example, APD reverse voltage VR or breakdown voltage VBR). Theprocessor then may output a value that is converted into an analogsignal. This signal may be used to influence the voltage at the feedbacknode of the flyback converter.

As will be further described, the second stage feedback may be generatedusing an attenuated output voltage read back through ananalog-to-digital converter (for example, 16-bit). This sameanalog-to-digital converter (or a separate analog-to-digital converter)may also be used to read in temperature data from the APD operatingenvironment.

FIG. 1 shows a flow cytometer 100, according to certain inventivetechniques. A stream of sperm cells 104 may pass through a flow chamber102, such that they may be single-file in certain strategic locations.The flow chamber may direct a fluid stream including the sperm cells 104(sample particles) through a particle interrogation location. The spermcells 104 may have been previously stained with a dye, such as aDNA-intercalating dye. Such a dye may fluoresce (or cause fluorescence)as it generates responsive light in response to being exposed to a light(or electromagnetic radiation) source. As the dyed sperm cells 104 passone-by-one, they may be exposed to a beam of electromagnetic radiation(for example, light of a given wavelength) generated by a laser andemitted along a beam path (and optionally associated optics 106) to theparticle interrogation location. Such associated optics may includelenses, filters, or the like. The exposure of the dye to the laser lightmay cause the dye to emit fluorescently generated light. The amount offluorescently generated light may vary by a detectable degree, dependingon whether the sperm cell 104 carries XX chromosomes or XY chromosomes.

The fluorescently generated light may be received by optics 108 (forexample, lenses, filters, or the like), and it may be focused onto anactive area of a photodetector 112. According to certain inventivetechniques, the photodetector 112 may include an APD. The photodetector112 may generate an output signal that corresponds (varies linearly ornon-linearly) to the amount of electromagnetic radiation (for example,light of a given wavelength) that it receives from the interrogationlocation. The photodetector output signal may include a time-varyinganalog signal indicative of an intensity of the received electromagneticradiation. This photodetector output signal may ultimately becommunicated to a processor 114 (which may include one processor or aplurality of processors that control a portion of the operation or theentire operation of the flow cytometer 100). The photodetector outputsignal may be amplified by at least one amplifier (or two or moreamplifiers in series) before it is communicated to the processor 114.Furthermore, the photodetector output signal (for example, as amplified)may be digitized before being communicated to the processor 114. Atemperature sensor 116 may generate an output signal that corresponds(varies linearly or non-linearly) to the sensed temperature. As such,the temperature signal may encode temperature data that corresponds to agiven sensed temperature. This temperature signal may ultimately becommunicated to the processor 114 (for example, after amplification).The processor 114 may also receive a signal corresponding to the biasvoltage (for example, the reverse-bias voltage) of the photodetector 112generated by the power supply 110 and filtered by the output filter 120.

It should be understood that the photodetector signal, the temperaturesignal, and the bias voltage signal may be conditioned and/or digitizedbefore they are received by the processor 114—that is, they need not bedirectly connected such that the exact voltages output by the sensors112, 116 or the output filter 120 are delivered to the processor 114.Instead, the system need only be designed such that the informationgenerated by the sensors 112, 116 and the bias voltage is communicatedto the processor 114. In this sense, these signals are communicated tothe processor 114.

Depending on the temperature signal, the bias voltage signal, and/orknown photodetector 112 characteristics, the processor 114 may influencethe voltage of the power supply 110 that conditions (for example,reverse-biases) the photodetector 112. Such known photodetector 112characteristics may include an APD reverse voltage VR or breakdownvoltage VBR. These characteristics may be stored in memory (not shown)which may be accessed by the processor 114 for computations. Such amemory may include a single memory or a plurality of memories separatelyaddressable. The memory may be within a package with the processor ormay be in a package external to the processor package. The memory may benon-volatile (for example, EEPROM or flash memory). The memory may alsostore relevant curves (for example, curves defining the input-outputrelationships of the photodetector 112 or the temperature sensor 116, orother characteristics within the flow cytometer 100).

The processor 114 may make a decision as to whether the sperm cell 104carries XX or XY chromosomes (that is, female or male gendered spermcells 104, respectively). If a sperm cell 104 does not carry the desiredset of chromosomes, the processor 114 may control the kill/segregationcomponentry 118 to kill or segregate the unwanted sperm cell 104. Inthis manner, a substantially high-purity population of gender-specificsperm cells 104 may be generated.

FIG. 2 shows a block diagram 200 for circuitry capable of detecting thechromosomal content of sperm cells using an avalanche photodiode(“APD”), according to certain inventive techniques. The block diagram200 may generally correspond to elements 110, 112, 114, 116, and 120depicted in FIG. 1 and discussed above. Furthermore, FIGS. 6A-6X depictcircuit schematics (just one exemplary embodiment of many possibilities)that correspond to block diagram 200, according to certain inventivetechniques.

Linear drop-out (“LDO”) regulator circuitry 202 (for example, includingan integrated circuit, such as Linear Technology's LT1764) may receivean input DC voltage and convert it to a regulated low voltage (e.g.,substantially 5 VDC). This block provides pre-regulation for the highvoltage that will be created downstream. This initial stage ofregulation may improve system noise by removing a substantial amount ofthe switching residue from the upstream voltage supply.

The regulated low voltage output from the LDO circuitry 202 is receivedby high-voltage generator circuitry 204, which generates a relativelyhigh voltage. Such circuitry 204 may include an integrated circuit (suchas Linear Technology's LT3580). The output of the high-voltage generatorcircuitry 204 may be provided to the transformer 206. The transformer206 may step the voltage up, for example, by a factor of 10. Schottkydiode(s) (one or more in series) may receive the output of thetransformer 206. The resulting voltage may be fed back to thehigh-voltage generator circuitry 204 through feedback circuitry 214 (forexample, one or more resistors in series).

The high-voltage generator circuitry 204, the transformer 206, and thefeedback circuitry 214 may form a power supply, such as a DC/DC boostswitching power supply. The power supply may be a boost configurationthat implements a resistive feedback circuit and the digital-to-analogconverter 220 to generate the output voltage (as will be furtherexplained).

Overall, the power supply may include input circuitry (for example,including the high-voltage generator circuitry 204), the transformer206, and output circuitry (for example, including one or more Schottkydiodes in series between the output of the transformer 206 and the firstfilter 208, or other suitable components). A feedback loop maycommunicate what the voltage is after the output circuitry to a feedbacknode arranged as an input to the high-voltage generator circuitry 204.

A first filter 208 may be located at the output of the power supply. Thefirst filter 208 may include a network of one or more capacitor(s)and/or resistor(s). For example, the first filter 208 may be an RC-typefilter. The first filter 208 may remove a degree of relativelyhigh-frequency noise (for example, switching noise). The output of thefirst filter 208 may be provided to APD circuitry 210. The filteredvoltage from the first filter 208 may be used to reverse-bias the APD,which may be included in the APD circuitry 210. The APD may include aHamamatsu S8664 series APD.

A second filter 212 may also be provided as part of the output filternetwork to the switching power supply. Altogether, the output filternetwork may include an RC-type filter (an example of the first filter208) followed by the APD circuitry 210, and an additional outputcapacitor used to provide additional device stabilization (an example ofthe second filter 212). Because the impedance of the APD mimics that ofa capacitor, it may be built into the output filter network and act as acomponent in the device output filter circuitry. The output filtercircuitry may use relatively large values that are typically too largefor commercially-practical power supply circuits. However, because theAPD requires relatively low bias currents, the output filter network canuse these otherwise impractical components to aid in achievingadvantageously low noise levels. For example, output voltage noisemeasured in a 20 MHz bandwidth has been shown to be below 150 microvolts(VRMS).

The APD circuitry 210 may output a current signal when it receives lightin a given frequency range. The current signal may be converted and/oramplifiers by one or more amplifiers. The amplifier(s) may include afirst amplifier 216 and optionally a second amplifier 218. The firstamplifier 216 may convert the current signal to a voltage signal. Thesecond amplifier 218 may provide additional gain if/as needed byamplifying the output provided by the first amplifier 216. The secondamplifier 218 may also invert the signal to mimic a PMT voltage outputsignal. Although not shown, the output of the second amplifier may be avoltage signal which may be digitized (for example, by A-to-D converter230) and communicated to a processor (such as processor 222). Adifferent processor (not shown) and/or another A-to-D converter (notshown) may be used for digitization/processing of the output signal. Inaddition to the feedback loop in the power supply, a second feedbackloop may be included in the circuitry represented by block diagram 200.This second feedback loop may include voltage adjustment circuitry,which is configured to adjust a voltage on the first feedback loop basedat least in part on a voltage measured between the first filter 208 andthe APD circuitry 210. This second feedback loop (including the voltageadjustment circuitry) may include readback circuitry 228, multiplexer226, A-to-D converter 230, processor 222, D-to-A converter 224, andcircuitry 220. The second feedback loop may influence the voltage at thefeedback node in the DC boost switching power supply to adjust itsoutput based potentially a variety of factors, including thesubstantially real-time voltage at or proximate the APD. The particulartechniques disclosed for the second feedback loop including voltageadjustment circuitry are just one of many different possible techniquesthat can influence the voltage at the feedback node of the power supplyaccording to a voltage measured between the first filter 208 and the APDcircuitry 210.

The readback circuitry 228 may receive the voltage between the firstfilter 208 and APD circuitry 210. The read back circuitry 228 mayinclude an amplifier to amplify the received voltage. The output of thereadback circuitry 228 may be provided to multiplexer 226. Themultiplexer 226 may be an analog multiplexer, and it may receive aplurality of input signals. Such signals may include the output of thereadback circuitry 228, the output of a temperature sensor amplifier232, and/or the output of the amplifier network that conditions theoutput signal from the APD circuitry 210 (not shown). The processor 222may provide a select signal to the multiplexer 226 to determine which ofthese (or other) signals will be output from the multiplexer 226. Theoutput of the multiplexer 226 is provided to the A-to-D converter 230(for example, 16-bit). The digitized output of the A-to-D converter 230may be provided to the processor 222. Thus, the digital signal encodesthe measured voltage between the first filter 208 and the APD circuitry210.

The processor 222 may execute an equation or algorithm (throughprocessing) to generate a digital output signal. The equation oralgorithm may account for different input variables, including thevoltage between the first filter 208 and the APD circuitry 210, thetemperature at or near the APD, and/or APD device characteristics (forexample, APD reverse voltage VR or breakdown voltage VBR). The outputsignal from the processor may be provided to the D-to-A converter 224(for example, 16-bit), and the analog output (an adjustment voltage) ofthe converter 224 may be received by circuitry 220. Circuitry 220 maycondition the signal (for example, filter/amplify the output of theconverter 224). The output of the circuitry 220 may influence or causethe voltage at the feedback input (or node) to the high-voltagegenerator circuitry 204 to change. The feedback node voltage isinfluenced by the electrical signal summation of the digital-to-analogconverter 224 voltage output and the output voltage of the DC-DCconverter. The feedback voltage generated from the output of the DC-DCconverter combines with the digital-to-analog converter 224 output togenerate the power supply output voltage.

The temperature sensor 234 (for example a TI LM35 series sensor) mayprovide a temperature reading with a minimum of 0.25° C. linearity. Thetemperature sensor 234 output may be amplified by temperature sensoramplifier 232 before it is provided to multiplexer 226. The temperaturemay be measured at periodic intervals for changes from when the lastuser adjustment from an operator occurred. Based on the change intemperature, the processor 222 may either increase or decrease the APDreverse-bias voltage. A slope calculation from the configuration step ofthe APD may be used to adjust the voltage bias by a fixed amount forevery 0.25° C. change at the temperature sensor (near or at the APD).

For example, each 0.25° C. temperature change may be converted into anumber of digital counts based on the slope of the output voltage. It isthen determined how many digital counts are needed need to generate ‘X’voltage value. The slope value is generated for each power supply bysetting the power supply's digital-to-analog converter 224, measuringthe output voltage of the power supply, setting the digital-to-analogconverter 224 to a different value, and measuring the output voltage.This results in an equation:(voltage2−voltage1)/(DAC_value2−DAC_value1)=slope. The slope informs howmuch voltage the output will change per digital-to-analog converter 224count value. It may be known that for every single degree C. change, thedevice will need to adjust by, for example, 0.78V. So for every 0.25°C., it may be known that the output will need to be adjusted by 0.78V/4,or 0.195V at the output. The slope determines how many digital countsare needed to effect the proper change (either up or down depending onif temperature is increasing or decreasing). For example, if the slopeis 0.0076, and the temperature changes by 0.25° C., thedigital-to-analog converter 224 may need to be adjusted by0.195V/0.0076, which equals 26 digital counts.

Consider the following illustrative example for the operation ofcircuitry illustrated by block diagram 200.

Each APD may have a different operating voltage bias and breakdownvoltage. The APD reverse voltage (VR) and breakdown voltage (VBR) may bespecified by the APD manufacturer. These characteristics of a particularAPD may be stored in memory (for example, non-volatile memory) readableby the processor (for example, integrated EEPROM). Thus, each cytometermay be individualized for a given APD. Supply output voltage values mayalso be measured (for example, using an external NIST traceablevoltmeter) as part of a one-time configuration process for eachcytometer, and these calibration values for the output voltage may thenstored in memory (along with the other unique variables for the APD asdiscussed above).

For each given system, the VREF value may control the APD's voltagebias. For example, a value of 0.5V at VREF may correspond to an opticalgain of M=50 for the APD, and moving the value from 0.1V to 1V mayadjust the gain of the APD by increasing the bias voltage. Protectionalgorithms may be built into the processor 222 such that an unreasonablyhigh VREF may not allow the APD to reach breakdown voltage, andtherefore the APD may be protected. Under certain conditions, becauseVREF adjusts the optical gain at the APD, this does not mean that thesupply simply adjusts the output voltage in the same increments for eachdevice for a given VREF. This is because from VREF=0.1 to 1V, thealgorithm used to control the flyback converter supply may treat theVREF signal as an optical gain parameter, and not a voltage adjustmentparameter. This may mean that a VREF of 0.5V may produce a differentvoltage value which is unique for the given APD installed in the system.If an APD has a VR of 403.3V for M=50, and a separate APD has a VR valueof 390.8V for M=50, the VREF input of 0.5V may still be selected toproduce the exact same optical gain of M=50. This means that the outputvoltage adjustment through VREF may actually be a function of thedesired optical gain, which may be unique to the APD device itself, andis not a uniformly applicable output voltage adjustment. Thesecustomized values corresponding to the characteristics may be programedinto each system at startup, and the customized characteristics mayenable uniform “black box” performance across all similarly configuredsystems. Because the flyback power supply may utilize a hybrid analogand digital feedback from different nodes, the output voltage to achievethe M=50 may be obtained by comparing the high voltage readback signalof the actual APD itself, and the processor 222 may generate an errorterm from the flyback's analog control generated value. The secondfeedback loop value may be converted into a digital count based on theslope of a linear curve fit performed at the initial startupconfiguration. When a user sets VREF at ostensibly 0.5V, the processor222 may recognize that based on the values entered, the particular APDinstalled needs a reverse-bias voltage of 403.3V, and sets the preciseVREF to be something potentially different from 0.5V.

The digital-to-analog converter 224 may be sent a value determined fromthe initial configuration at startup, and, if the APD was not present inthe output filter, it may have the correct output voltage. However,because the first filter 208 may use relatively large value resistorcomponent(s), the feedback network of the supply may “think” it isproviding the correct voltage when really it is offset because thesystem now has an APD present in the output filter network. The secondfeedback node, which may be a digitized value of the actual outputvoltage at or proximate the APD (for example, a voltage at a locationbetween the first filter 208 and the APD circuitry 210), may then beused to solve an internal slope compensation equation (or other suitablealgorithm) in the processor 222 that sets the correct output voltage fora desired optical gain. This algorithm may then be used to adjust theD-to-A converter 224 value sent to the flyback converter's feedbacknode.

Temperature compensation may also be implemented. Once the optical gainis set for M=50, the processor may measure the temperature, and forexample, for any changes in 0.5° C. (or other suitable increment), theoutput voltage that was used for M=50 may be adjusted to a new value,for example, based on the breakdown coefficient of the APD to keep thegain set at M=50. This means that temperature compensation may beinvoked after an interval of time has passed since VREF has changed.This may allow for temperature once a user has set VREF (because not allsystems will want M=50, some may be at M=55, M=60, or other suitableoptical gains depending on the desired mode of operation).

FIG. 3 illustrates a flowchart 300 for a method, according to certaininventive techniques. The method may be performed by systems 100 or 200.The certain steps in the flowchart 300 may be performed at timesoverlapping other steps or substantially simultaneously with othersteps. Certain steps could be performed in a different order, and thereis no implication by the flow of flowchart 300 that steps must beperformed in any particular order.

At step 302, an input voltage may be received at input circuitry (forexample, high-voltage generator circuitry 204) of a power supply, suchas a DC/DC boost switching power supply. At step 304, a transformer (forexample, transformer 206) transforms a voltage supplied by the inputcircuitry into a transformed voltage. At step 306, output circuitry (forexample, one or more Schottky diodes in series between the transformer206 and the first filter 208) of the power supply receives thetransformed voltage. At step 308, the output circuitry generates anoutput voltage. At step 310, a voltage corresponding to the outputvoltage is fed back to the input circuitry. At step 312, the outputvoltage is received at a filter (for example, first filter 208), and afiltered voltage is generated by the filter. At step 314, the filteredvoltage is received at APD circuitry (for example, APD circuitry 210),which includes an APD. At step 316, the filtered voltage is received atvoltage adjustment circuitry (for example, readback circuitry 228). Thiscircuitry may be part of the second feedback loop, as discussed above.At step 318, the feedback voltage may be adjusted by the voltageadjustment circuitry according to at least the output voltage.

At step 320, a temperature sensor (for example, temperature sensor 234)may generate a temperature signal corresponding to a measuredtemperature (that is, a sensed temperature). Furthermore, step 318 mayfurther include adjusting the feedback voltage by the voltage adjustmentcircuitry according to at least the filtered voltage and the measuredtemperature. Additionally, step 318 may further adjusting the feedbackvoltage by the voltage adjustment circuitry according to at least thefiltered voltage, the measured temperature, and at least one valuecorresponding to a characteristic of the avalanche photodiode. Also,step 318 may further include adjusting the feedback voltage by thevoltage adjustment circuitry according to at least the filtered voltageand at least one value corresponding to a characteristic of theavalanche photodiode.

According to one technique, step 318 further includes: converting, withan analog-to-digital converter (for example, analog-to-digital converter230), the filtered voltage into a digital measured signal encodingfiltered voltage data; processing, with a processor (for example,processor 222), at least the filtered voltage data to generate a digitaladjustment signal; converting, by a digital-to-analog converter (forexample, digital-to-analog converter 224), the digital adjustment signalto an adjustment voltage; and adjusting the feedback voltage accordingto the adjustment voltage. According to another technique, saidprocessing at least the output voltage data may further includeprocessing at least temperature data and the filtered voltage data togenerate the digital adjustment signal. According to another technique,said processing at least the output voltage data further comprisesprocessing at least data corresponding to at least one characteristic ofthe avalanche photodiode, temperature data, and the filtered voltagedata to generate the digital adjustment signal. According to anothertechnique, the at least one characteristic of the avalanche photodiodecomprises at least one of a breakdown voltage and a reverse bias voltagecorresponding to a predetermined optical gain.

At step 322, a first amplifier (for example, first amplifier 216) mayamplify a voltage at an anode of the avalanche photodiode to form afirst amplified voltage. At step 324, a second amplifier (for example,second amplifier 218) may amplify the first amplified voltage togenerate a second amplified voltage.

FIG. 4 illustrates a flowchart 400 for a method, according to certaininventive techniques. The method may be performed by systems 100 or 200.The certain steps in the flowchart 400 may be performed at timesoverlapping other steps or substantially simultaneously with othersteps. Certain steps could be performed in a different order, and thereis no implication by the flow of flowchart 400 that steps must beperformed in any particular order.

At step 402, electromagnetic radiation (for example, light of a givenwavelength) is emitted from a laser (for example, laser 106). At step404, a fluid stream and sperm cells contained therein are illuminatedwith the electromagnetic radiation. At step 406, an APD (for example,photodetector 112) detects electromagnetic radiation emitted from thesperm cells. This emitted radiation (for example, light of a givenwavelength) may be generated by a fluorescing dye (for example, stainedon sperm chromosomes) that generates responsive radiation in response toreceived radiation. At step 408, the APD generates a time-varying analogsignal indicative of an intensity of the detected electromagneticradiation. At step 410, an analog-to-digital converter (for example,converter 230) converts the time-varying analog signal into acorresponding digital signal. The time-varying signal may be processedbefore digitization (for example, by readback circuitry 228). At step412, a processor (for example, processor 222) analyzes the digitalsignal to determine characteristics of the sperm cells in the fluidstream.

FIG. 5 illustrates a flowchart 500 for a method, according to certaininventive techniques. The method may be performed by systems 100 or 200.The certain steps in the flowchart 500 may be performed at timesoverlapping other steps or substantially simultaneously with othersteps. Certain steps could be performed in a different order, and thereis no implication by the flow of flowchart 500 that steps must beperformed in any particular order.

At step 502, DNA within a nucleus of a sperm cell (for example, spermcell 104) is stained, for example, with a DNA-intercalating, fluorescingdye. At step 504, the stained DNA within the nucleus of the sperm cellis irradiated (for example, by laser 106). At step 506, fluorescentlight emitted from the irradiated and stained DNA within the nucleus ofthe sperm cell is detected with an avalanche photodiode (for example,photodetector 112).

At step 508, a sex of a sperm cell is determined using the detectedamount of DNA within the nucleus of the sperm cell. For example, a spermcell with XX chromosomes may have 3% more DNA than a sperm cell with XYchromosomes. This may lead to a corresponding increase (linear ornon-linear increase) of light emission from the stained DNA. Due to theinventive techniques disclosed herein, it may be possible to measurethis difference with an avalanche photodiode, thereby determining the“sex” of a sperm cell. At step 510, a plurality of sperm cells aredifferentiated based upon said sex determination. The cells may besorted (creating two or more populations; for example, an X-chromosomepopulation and a non-X-chromosome population), or cells within thepopulation can be selected for deactivation (for example, by laserablation).

At step 512, a given sperm cell may be deactivated based upon thedetermined amount of DNA within the nucleus of the given sperm cell. Forexample, processor 114 may control operation of kill/segregationcomponentry 118 to deactivate (separate, degrade, or destroy) the spermcell so it may not be useful for fertilization based on the result ofstep 510. One technique of deactivation is illustrated generally bysteps 514, 516, 518, and 520. At step 514, a plurality of droplets maybe formed for entraining a corresponding plurality of the sperm cells.At step 516, each of the plurality of droplets may be differentiallycharged based upon the sex differentiation characteristic of thecorresponding entrained sperm cells. At step 518, each of the pluralityof droplets may be deflected. At step 520, each of the droplets may bedifferentially collected based upon the sex differentiationcharacteristic of the plurality of sperm cells entrained in thecorresponding plurality of droplets.

For example, one approach for sexing is laser-kill in which anythingthat is not an X-chromosome-bearing sperm cell may be ablated via laserpulse. Such techniques are described in U.S. Pat. Nos. 8,933,395,9,000,357, 9,140,690, and 9,335,295, the entireties of which are hereinincorporated by reference.

As another example, charge/deflection techniques are also used in thesexing industry. Such techniques are described in U.S. Pat. No.9,145,590, the entirety of which is herein incorporated by reference.Other conceivable approaches include the use of optical traps and/orlaser steering, as described in U.S. Pat. Nos. 8,149,416 and 8,158,927,the entireties of which are herein incorporated by reference.

It will be understood by those skilled in the art that various changesmay be made and equivalents may be substituted without departing fromthe scope of the novel techniques disclosed in this application. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the novel techniques without departingfrom its scope. Therefore, it is intended that the novel techniques notbe limited to the particular techniques disclosed, but that they willinclude all techniques falling within the scope of the appended claims.

What is claimed is:
 1. A flow cytometry apparatus comprising: a flowchamber configured to direct a fluid stream including cells through aninterrogation location; a laser configured to emit electromagneticradiation along a beam path to the interrogation location to illuminatea given cell; at least one avalanche photodiode configured to: receiveelectromagnetic radiation from the interrogation location emitted by thegiven cell; and generate a signal indicative of an intensity of thereceived electromagnetic radiation; at least one processor configured toanalyze the signal indicative of the intensity of the receivedelectromagnetic radiation to determine a characteristic of the givencell; a switching power supply including a feedback loop; a temperaturesensor configured to sense a temperature and generate a correspondingtemperature signal encoding temperature data; and voltage adjustmentcircuitry configured to adjust a voltage on the feedback loop based atleast in part on the temperature data.
 2. The flow cytometry apparatusof claim 1, wherein the characteristic of the given cell comprises anamount of chromosomes.
 3. The flow cytometry apparatus of claim 1,wherein the characteristic of the given cell comprises an amount of DNA.4. The flow cytometry apparatus of claim 1, wherein the characteristicof the given cell comprises a sex-differentiation characteristic.
 5. Theflow cytometry apparatus of claim 1, further comprising a filterelectrically connected between the switching power supply and the atleast one avalanche photodiode, wherein the voltage adjustment circuitryis further configured to adjust the voltage on the feedback loop basedat least in part on a voltage measured between the filter and the atleast one avalanche photodiode.
 6. The flow cytometry apparatus of claim5, wherein the voltage adjustment circuitry is further configured toadjust the voltage on the feedback loop based at least in part on atleast one characteristic of the at least one avalanche photodiode. 7.The flow cytometry apparatus of claim 5, wherein the voltage adjustmentcircuitry comprises: an analog-to-digital converter configured toconvert the voltage measured between the filter and the at least oneavalanche photodiode into a digital measured signal encoding measuredvoltage data; the at least one processor configured to process at leastthe measured voltage data to generate a digital adjustment signal; and adigital-to-analog converter configured to convert the digital adjustmentsignal to an adjustment voltage, wherein the adjustment voltageinfluences the voltage on the feedback loop.
 8. The flow cytometryapparatus of claim 7, wherein the processor is configured to process atleast the temperature data and the measured voltage data to generate thedigital adjustment signal.
 9. The flow cytometry apparatus of claim 7,wherein the processor is configured to process at least temperaturedata, data corresponding to at least one characteristic of the avalanchephotodiode, and the measured voltage data to generate the digitaladjustment signal.
 10. The flow cytometry apparatus of claim 9, whereinthe at least one characteristic of the at least one avalanche photodiodecomprises at least one of a breakdown voltage and a reverse bias voltagecorresponding to a predetermined optical gain.
 11. The flow cytometryapparatus of claim 5, wherein the switching power supply comprises aDC/DC boost or flyback power supply.
 12. The flow cytometry apparatus ofclaim 1, wherein: the at least one avalanche photodiode is arranged toreceive an amount of fluorescent light emitted by each of the cells; theamount of received fluorescent light varies amongst the cells based atleast in part upon a relative amount of at least one particledifferentiation characteristic present in each of the cells; and whereinthe at least one avalanche photodiode is configured to convert theamount of received fluorescent light into at least one signal whichvaries based upon the amount of received fluorescent light.
 13. The flowcytometry apparatus of claim 1, further comprising kill componentrycontrolled by the at least one processor to kill the given cellaccording to the characteristic of the given cell.
 14. The flowcytometry apparatus of claim 1, further comprising segregationcomponentry controlled by the at least one processor to segregate thegiven cell according to the characteristic of the given cell.
 15. A flowcytometry apparatus comprising: a flow chamber configured to direct afluid stream including cells through an interrogation location; a laserconfigured to emit electromagnetic radiation along a beam path to theinterrogation location to illuminate a given cell; at least oneavalanche photodiode configured to: receive electromagnetic radiationfrom the interrogation location emitted by the given cell; and generatea signal indicative of an intensity of the received electromagneticradiation; at least one processor configured to analyze the signalindicative of the intensity of the received electromagnetic radiation todetermine a characteristic of the given cell; and circuitry configuredto deliver a signal indicative of the intensity of the receivedelectromagnetic radiation in a digital form to the at least oneprocessor, wherein the circuitry is interposed between the at least oneavalanche photodiode and the at least one processor and further whereinthe circuitry comprises: a first amplifier configured to receive in ananalog form, amplify, and output the signal indicative of the intensityof the received electromagnetic radiation from the at least oneavalanche photodiode; a second amplifier configured to receive in theanalog form, amplify, and invert the output of the first amplifier; andat least one analog-to-digital converter configured to convert thesignal indicative of the intensity of the received electromagneticradiation into the digital form.
 16. A method of using a flow cytometryapparatus to differentiate characteristics of cells comprising:receiving an input voltage at input circuitry of a power supply;transforming, with a transformer, a voltage supplied by the inputcircuitry into a transformed voltage; receiving, at output circuitry ofthe power supply, the transformed voltage; generating, by the outputcircuitry, an output voltage; feeding back a feedback voltagecorresponding to the output voltage to the input circuitry; receivingthe output voltage at a filter; generating, by the filter, a filteredvoltage; receiving the filtered voltage at at least one avalanchephotodiode; receiving the filtered voltage at voltage adjustmentcircuitry; and adjusting the feedback voltage by the voltage adjustmentcircuitry according to at least the filtered voltage.
 17. The method ofclaim 16, further comprising: generating, by a temperature sensor, atemperature signal corresponding to a measured temperature, wherein saidadjusting the feedback voltage further comprises adjusting the feedbackvoltage by the voltage adjustment circuitry according to at least thefiltered voltage and the measured temperature.
 18. The method of claim16, wherein said adjusting the feedback voltage by the voltageadjustment circuitry is according to at least the filtered voltage andat least one value corresponding to a characteristic of at least oneavalanche photodiode.
 19. The method of claim 17, further comprising:adjusting said feedback voltage by the voltage adjustment circuitryaccording to at least the filtered voltage, the measured temperature,and at least one value corresponding to a characteristic of at least oneavalanche photodiode.
 20. The method of claim 16, further comprising:converting, with an analog-to-digital converter, said filtered voltageinto a digital measured signal encoding filtered voltage data;processing, with a processor, at least the said filtered voltage data togenerate a digital adjustment signal; converting, by a digital-to-analogconverter, the digital adjustment signal to an adjustment voltage; andadjusting the feedback voltage according to the adjustment voltage. 21.The method of claim 20, further comprising: generating, by a temperaturesensor, a temperature signal corresponding to a measured temperature,wherein said adjusting the feedback voltage further comprises adjustingthe feedback voltage by the voltage adjustment circuitry according to atleast the filtered voltage and the measured temperature.
 22. The methodof claim 21, further comprising processing at least temperature data andthe filtered voltage data to generate said digital adjustment signal.23. The method of claim 21, further comprising processing at least datacorresponding to at least one characteristic of at least one avalanchephotodiode, temperature data, and the filtered voltage data to generatesaid digital adjustment signal.
 24. The method of claim 21, wherein saidat least one characteristic of at least one avalanche photodiodeincludes at least one of a breakdown voltage and a reverse bias voltagecorresponding to a predetermined optical gain.
 25. The method of claim16, further comprising: amplifying, with a first amplifier, a voltage atan anode of at least one avalanche photodiode to form a first amplifiedvoltage; and amplifying, with a second amplifier, the first amplifiedvoltage to generate a second amplified voltage.
 26. A method foranalyzing sperm cells contained in a fluid stream as the sperm cellsflow through an interrogation location comprising: emittingelectromagnetic radiation from a laser; illuminating, with theelectromagnetic radiation emitted by the laser, the fluid stream and thesperm cells contained therein; detecting, with an avalanche photodiode,electromagnetic radiation emitted from the interrogation location;generating, by at least one avalanche photodiode, a time-varying analogsignal indicative of an intensity of the detected electromagneticradiation; converting, with an analog-to-digital converter, thetime-varying analog signal into corresponding digital signal; andanalyzing, by a processor, the digital signal to determinecharacteristics of the sperm cells in the fluid stream.
 27. A method forassessing an amount of DNA within a nucleus of a sperm cell comprising:staining the DNA within the nucleus of the sperm cell; irradiating thestained DNA within the nucleus of the sperm cell; and detecting, with atleast one avalanche photodiode, fluorescent light emitted from theirradiated and stained DNA within the nucleus of the sperm cell.
 28. Themethod of claim 27, further comprising: determining a sex of a spermcell using the detected fluorescent light within the nucleus of thesperm cell; and differentiating between a plurality of sperm cells basedupon said sex determination.
 29. The method of claim 28, furthercomprising: deactivating a sperm cell based upon the determined amountof DNA within the nucleus of said sperm cell.
 30. The method of claim29, further comprising photo-damaging said sperm cell.
 31. The method ofclaim 28 further comprising: forming droplets a plurality having one ofsaid sperm cells entrained; charging each of said dropletsdifferentially based upon said sex differentiation characteristic ofsaid sperm cells entrained in said droplets; deflecting each of saiddroplets; and differentially collecting each of said droplets based uponsaid sex differentiation characteristic of said sperm cells entrained insaid droplets.